1. Field of the Invention
The present invention is in the fields of protein biochemistry and the pharmaceutical and medical sciences. In particular, the invention provides methods for the production of conjugates between water-soluble polymers (e.g., poly(ethylene glycol) and derivatives thereof) and cytokines (e.g., interferon-beta), which conjugates have increased potency compared to polymer conjugates of the same cytokine synthesized by standard methods. The invention also provides conjugates produced by such methods, compositions comprising such conjugates, kits comprising such conjugates and compositions and methods of use of the conjugates and compositions in preventing, diagnosing and treating a variety of medical and veterinary conditions. The invention also provides methods of determining the site(s) of attachment of polymers by reductive alkylation under certain conditions.
2. Related Art
The following description of related art includes interpretations of the present inventors that are not, themselves, in the prior art. Cytokines are secreted regulatory proteins that control the survival, growth, differentiation, and/or effector function of cells in endocrine, paracrine or autocrine fashion (reviewed in Nicola, N. A. (1994) in: Guidebook to Cytokines and Their Receptors, Nicola, N. A., ed., pp. 1-7, Oxford University Press, New York). Because of their potency, specificity, small size and relative ease of production in recombinant organisms, cytokines have many potential applications as therapeutic agents. Two key factors have hindered the development of cytokines, in particular, and recombinant proteins, in general, as therapeutic agents—their generally short half-lives in the circulation and their potential antigenicity and immunogenicity. As used herein and generally in the art, the term “antigenicity” refers to the ability of a molecule to bind to preexisting antibodies, while the term “immunogenicity” refers to the ability of the molecule to evoke an immune response in vivo, whether that response involves the formation of antibodies (a “humoral response”) or the stimulation of cellular immune responses.
For the administration of recombinant therapeutic proteins, intravenous (i.v.) administration is often desirable in order to achieve the highest circulating activities and to minimize problems of bioavailability and degradation. However, the half-lives of small proteins following i.v. administration are usually extremely short (see examples in Mordenti, J., et al., (1991) Pharm Res 8:1351-1359; Kuwabara, T., et al., (1995) Pharm Res 12:1466-1469). Proteins with hydrodynamic radii exceeding that of serum albumin, which has a Stokes radius of about 36 Å and a molecular weight of about 66,000 Daltons (66 kDa), are generally retained in the bloodstream by healthy kidneys. However, smaller proteins, including cytokines such as granulocyte colony-stimulating factor (“G-CSF”), interleukin-2 (“IL-2”), interferon-alpha (“IFN-alpha”) and interferon-gamma (“IFN-gamma”), are cleared rapidly from the bloodstream by glomerular filtration (Brenner, B. M., et al., (1978) Am J Physiol 234:F455-F460; Venkatachalam, M. A. et al., (1978) Circ Res 43:337-347; Wilson, G., (1979) J Gen Physiol 74:495-509; Knauf, M. J., et al., (1988) J Biol Chem 263:15064-15070; Kita, Y., et al., (1990) Drug Des Deliv 6:157-167; Rostaing, L., et al., (1998), J Am Soc Nephrol 9:2344-2348). As a result, the maintenance of therapeutically useful concentrations of small recombinant proteins in the circulation is problematic following injection. Therefore, higher concentrations of such proteins and more frequent injections typically must be administered. The resulting dose regimens increase the cost of therapy, decrease the likelihood of patient compliance and increase the risk of adverse events, e.g., immune reactions. Both cellular and humoral immune responses can reduce the circulating concentrations of injected recombinant proteins to an extent that may preclude the administration of an effective dose or may lead to treatment-limiting events including accelerated clearance, neutralization of efficacy and anaphylaxis (Ragnhammar, P., et al., (1994) Blood 84:4078-4087; Wadhwa, M., et al., (1999) Clin Cancer Res 5:1353-1361; Hjelm Skog, A.- L., et al., (2001) Clin Cancer Res 7:1163-1170; Li, J., et al., (2001) Blood 98:3241-3248; Basser, R. L., et al., (2002) Blood 99:2599-2602; Schellekens, H., (2002) Clin Ther 24:1720-1740).
Modification of recombinant proteins by the covalent attachment of poly(ethylene glycol) (“PEG”) has been investigated extensively as a means of addressing the shortcomings discussed above (reviewed in Sherman, M. R., et al., (1997) in: Poly(ethylene glycol): Chemistry and Biological Applications, Harris, J. M., et al., eds., pp. 155-169, American Chemical Society, Washington, D.C.; Roberts, M. J., et al., (2002) Adv Drug Deliv Rev 54:459-476). The attachment of PEG to proteins has been shown to stabilize the proteins, improve their bioavailability and/or reduce their immunogenicity in vivo. (The covalent attachment of PEG to a protein or other substrate is referred to herein, and is known in the art, as “PEGylation.”) In addition, PEGylation can increase the hydrodynamic radius of proteins significantly. When a small protein such as a cytokine is coupled to a single long strand of PEG (e.g., having a molecular weight of at least about 18 kDa), the resultant conjugate has a hydrodynamic radius exceeding that of serum albumin and its clearance from the circulation via the renal glomeruli is retarded dramatically. The combined effects of PEGylation—reduced proteolysis, reduced immune recognition and reduced rates of renal clearance—confer substantial advantages on PEGylated proteins as therapeutic agents.
Since the 1970s, attempts have been made to use the covalent attachment of polymers to improve the safety and efficacy of various proteins for pharmaceutical use (see, e.g., Davis, F. F., et al., U.S. Pat. No. 4,179,337). Some examples include the coupling of PEG or poly(ethylene oxide) (“PEO”) to adenosine deaminase (EC 3.5.4.4) for use in the treatment of severe combined immunodeficiency disease (Davis, S., et al., (1981) Clin Exp Immunol 46:649-652; Hershfield, M. S., et al., (1987) N Engl J Med 316:589-596), to superoxide dismutase (EC 1.15.1.1) for the treatment of inflammatory conditions (Saifer, M., et al., U.S. Pat. Nos. 5,006,333 and 5,080,891) and to urate oxidase (EC 1.7.3.3) for the elimination of excess uric acid from the blood and urine (Kelly, S. J., et al., (2001) J Am Soc Nephrol 12:1001-1009; Williams, L. D., et al., PCT Publication No. WO 00/07629 A3 and U.S. Pat. No. 6,576,235; Sherman, M. R., et al., PCT Publication No. WO 01/59078 A2).
PEOs and PEGs are polymers composed of covalently linked ethylene oxide units. These polymers have the following general structure:R1—(OCH2CH2)n—R2 where R2 may be a hydroxyl group (or a reactive derivative thereof) and R1 may be hydrogen, as in dihydroxyPEG (“PEG diol”), a methyl group, as in monomethoxyPEG (“mPEG”), or another lower alkyl group, e.g., as in iso-propoxyPEG or t-butoxyPEG. The parameter n in the general structure of PEG indicates the number of ethylene oxide units in the polymer and is referred to herein and in the art as the “degree of polymerization.” Polymers of the same general structure, in which R1 is a C1-7 alkyl group, have also been referred to as oxirane derivatives (Yasukohchi, T., et al., U.S. Pat. No. 6,455,639). PEGs and PEOs can be linear, branched (Fuke, I., et al., (1994) J Control Release 30:27-34) or star-shaped (Merrill, E. W., (1993) J Biomater Sci Polym Ed 5: 1-11). PEGs and PEOs are amphipathic, i.e., they are soluble in water and in certain organic solvents and they can adhere to lipid-containing materials, including enveloped viruses and the membranes of animal and bacterial cells. Certain random or block or alternating copolymers of ethylene oxide (OCH2CH2) and propylene oxide, which has the following structure:
have properties that are sufficiently similar to those of PEG that these copolymers are thought to be suitable replacements for PEG in certain applications (see, e.g., Hiratani, H., U.S. Pat. No. 4,609,546 and Saifer, M., et al., U.S. Pat. No. 5,283,317). The term “polyalkylene oxides” and the abbreviation “PAOs” are used herein to refer to such copolymers, as well as to PEG or PEO and poly(oxyethylene-oxymethylene) copolymers (Pitt, C. G., et al., U.S. Pat. No. 5,476,653). As used herein, the term “polyalkylene glycols” and the abbreviation “PAGs” are used to refer generically to polymers suitable for use in the conjugates of the invention, particularly PEGs, more particularly PEGs containing a single reactive group (“monofunctionally activated PEGs”).
The covalent attachment of PEG or other polyalkylene oxides to a protein requires the conversion of at least one end group of the polymer into a reactive functional group. This process is frequently referred to as “activation” and the product is called “activated PEG” or activated polyalkylene oxide. MonomethoxyPEGs, in which an oxygen at one end is capped with an unreactive, chemically stable methyl group (to produce a “methoxyl group”) and on the other end with a functional group that is reactive towards amino groups on a protein molecule, are used most commonly for such approaches. So-called “branched” mPEGs, which contain two or more methoxyl groups distal to a single activated functional group, are used less commonly. An example of branched PEG is di-mPEG-lysine, in which PEG is coupled to both amino groups, and the carboxyl group of lysine is most often activated by esterification with N-hydroxysuccinimide (Martinez, A., et al., U.S. Pat. No. 5,643,575; Greenwald, R. B., et al., U.S. Pat. No. 5,919,455; Harris, J. M., et al., U.S. Pat. No. 5,932,462).
Commonly, the activated polymers are reacted with a bioactive compound having nucleophilic functional groups that serve as attachment sites. One nucleophilic functional group that is used commonly as an attachment site is the epsilon amino group of lysine residues. Solvent-accessible alpha-amino groups, carboxylic acid groups, guanidino groups, imidazole groups, suitably activated carbonyl groups, oxidized carbohydrate moieties and thiol groups have also been used as attachment sites.
The hydroxyl group of PEG has been activated with cyanuric chloride prior to its attachment to proteins (Abuchowski, A., et al., (1977) J Biol Chem 252:3582-3586; Abuchowski, A., et al., (1981) Cancer Treat Rep 65:1077-1081). The use of this method has disadvantages, however, such as the toxicity of cyanuric chloride and its non-specific reactivity for proteins having functional groups other than amines, such as solvent-accessible cysteine or tyrosine residues that may be essential for function. In order to overcome these and other disadvantages, alternative activated PEGs have been introduced, such as succinimidyl succinate derivatives of PEG (“SS-PEG”) (Abuchowski, A., et al., (1984) Cancer Biochem Biophys 7:175-186), succinimidyl carbonate derivatives of PAG (“SC-PAG”) (Saifer, M., et al., U.S. Pat. No. 5,006,333) and aldehyde derivatives of PEG (Royer, G. P., U.S. Pat. No. 4,002,531).
Commonly, several (e.g., 5 to 10) strands of one or more PAGs, e.g., one or more PEGs with a molecular weight of about 5 kDa to about 10 kDa, are coupled to the target protein via primary amino groups (the epsilon amino groups of lysine residues and, possibly, the alpha amino group of the amino-terminal (“N-terminal”) amino acid). More recently, conjugates have been synthesized containing a single strand of mPEG of higher molecular weight, e.g., 12 kDa, 20 kDa or 30 kDa. Direct correlations have been demonstrated between the plasma half-lives of the conjugates and an increasing molecular weight and/or increasing number of strands of PEG coupled (Knauf, M. J., et al., supra; Katre, N. V. (1990) J Immunol 144:209-213; Clark, R., et al., (1996) J Biol Chem 271:21969-21977; Bowen, S., et al., (1999) Exp Hematol 27:425-432; Leong, S. R., et al., (2001) Cytokine 16:106-119). On the other hand, as the number of strands of PEG coupled to each molecule of protein is increased, so is the probability that an amino group in an essential region of the protein will be modified and hence the biological function of the protein will be impaired, particularly if it is a receptor-binding protein. For larger proteins that contain many amino groups, and for enzymes with substrates of low molecular weight, the tradeoff between increased duration of action and decreased specific activity may be acceptable, since it produces a net increase in the biological activity of the PEG-containing conjugates in vivo. For smaller proteins that function via interactions with cell-surface receptors, such as cytokines, however, a relatively high degree of substitution has been reported to decrease the functional activity to the point of negating the advantage of an extended half-life in the bloodstream (Clark, R., et al., supra).
Thus, polymer conjugation is a well-established technology for prolonging the bioactivity and decreasing the immunoreactivity of therapeutic proteins such as enzymes (see, e.g., U.S. Provisional Appl. No. 60/436,020, filed Dec. 26, 2002, and U.S. Provisional Appl. Nos. 60/479,913 and 60/479,914, both filed on Jun. 20, 2003, the disclosures of which are incorporated herein by reference in their entireties). A class of therapeutic proteins that would benefit especially from such decreased immunoreactivity are the interferon-betas, particularly interferon-beta-1b (“IFN-β-1b;” SEQ ID NO: 1) (The IFNB Multiple Sclerosis Study Group (1996) Neurology 47:889-894). However, the conjugation of polymers to regulatory proteins that function by binding specifically to cell-surface receptors usually: (1) interferes with such binding; (2) markedly diminishes the signal transduction potencies of cytokine agonists; and (3) markedly diminishes the competitive potencies of cytokine antagonists. Published examples of such conjugates with diminished receptor-binding activity include polymer conjugates of granulocyte colony-stimulating factor (“G-CSF”) (Kinstler, O., et al., PCT Publication No. WO 96/11953; Bowen, S., et al., supra); human growth hormone (“hGH”) (Clark, R., et al., supra); hGH antagonists (Ross, R. J. M., et al., (2001) J Clin Endocrinol Metab 86:1716-1723; and IFN-alpha (Bailon, P., et al., (2001) Bioconjug Chem 12:195-202; Wylie, D. C., et al., (2001) Pharm Res 18:1354-1360; and Wang, Y.- S., et al., (2002) Adv Drug Deliv Rev 54:547-570), among others. In an extreme case, the coupling of polymers to interleukin-15 (“IL-15”) converted this IL-2-like growth factor into an inhibitor of cellular proliferation (Pettit, D. K., et al., (1997) J Biol Chem 272:2312-2318). While not intending to be bound by theory, the mechanism for such undesirable effects of PEGylation may involve steric hindrance of receptor interactions by the bulky PEG groups, charge neutralization, or both.
Thus, there exists a need for methods for producing PAG-containing (e.g., PEG- and/or PEO-containing) conjugates, particularly conjugates between such water-soluble polymers and receptor-binding proteins, with preservation of substantial bioactivity (e.g., at least about 40%), nearly complete bioactivity (e.g., at least about 80%) or essentially complete bioactivity (e.g., at least about 90%). Such conjugates will have the benefits provided by the polymer component of increased solubility, stability and bioavailability in vivo and will exhibit substantially increased potency or utility, compared to conventional polymer conjugates, in an animal into which the conjugates have been introduced for prophylactic, therapeutic or diagnostic purposes.